Methylation assay

ABSTRACT

A method for detecting a methylated genomic locus is provided. In certain embodiments, the method comprises: a) treating a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an agent that modifies cytosine to uracil to produce a treated nucleic acid; b) amplifying a product from the treated nucleic acid using a first primer and a second primer, wherein the first primer hybridizes to a site in the locus that contain methylcytosines and the amplifying preferentially amplifies the methylated copies of the genomic locus, to produce an amplified sample; and c) detecting the presence of amplified methylated copies of the genomic locus in the amplified sample using a flap assay that employs an invasive oligonucleotide having a 3′ terminal G or C nucleotide that corresponds to a site of methylation in the genomic locus.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/170,541, filed Feb. 8, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/522,500, filed on Jul. 25, 2019, which is acontinuation of U.S. patent application Ser. No. 14/539,841, filed Nov.12, 2014, which is a continuation of U.S. patent application Ser. No.12/946,745, filed Nov. 15, 2010, now U.S. Pat. No. 8,916,344, issuedDec. 23, 2014.

BACKGROUND

The methylation of cytosine residues in DNA is an important epigeneticalteration in eukaryotes. In humans and other mammals methylcytosine isfound almost exclusively in cytosine-guanine (CpG) dinucleotides. DNAmethylation plays an important role in gene regulation and changes inmethylation patterns are involved in human cancers and certain humandiseases. Among the earliest and most common genetic alterationsobserved in human malignancies is the aberrant methylation of CpGislands, particularly CpG islands located within the 5′ regulatoryregions of genes, causing alterations in the expression of such genes.Consequently, there is great interest in using DNA methylation markersas diagnostic indicators for early detection, risk assessment,therapeutic evaluation, recurrence monitoring, and the like. There isalso great scientific interest in DNA methylation for studyingembryogenesis, cellular differentiation, transgene expression,transcriptional regulation, and maintenance methylation, among otherthings.

This disclosure relates to the detection of methylated DNA in a sample.

Incorporation by Reference of Sequence Listing Provided as a SequenceListing Xml File

A Sequence Listing is provided herewith as a Sequence Listing XML,“EXAS-002CON4_SEQ_LIST”, created on May 12, 2023 and having a size of103,893 bytes. The contents of the Sequence Listing XML are incorporatedherein by reference in their entirety.

SUMMARY

A method for detecting a methylated genomic locus is provided. Incertain embodiments, the method comprises: a) treating a nucleic acidsample that contains both unmethylated and methylated copies of agenomic locus with an agent that modifies cytosine to uracil to producea treated nucleic acid; b) amplifying a product from the treated nucleicacid using a first primer and a second primer, wherein the first primerhybridizes to a site in the locus that contain methylcytosines and theamplifying preferentially amplifies the methylated copies of the genomiclocus, to produce an amplified sample; and c) detecting the presence ofamplified methylated copies of the genomic locus in the amplified sampleusing a flap assay that employs an invasive oligonucleotide having a 3′terminal G or C nucleotide that corresponds to a site of methylation inthe genomic locus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates some of the general principles of aflap assay.

FIG. 2 schematically illustrates one embodiment of the subject method.

FIG. 3 show the nucleotide sequences of methylated and unmethylatedcopies of a fragment of the human vimentin gene (VIM), before and afterbisulfite treatment.

FIG. 4 shows the nucleotide sequences of an exemplary forward primer, anexemplary reverse primer, and an exemplary flap oligonucleotide, alignedwith the fragments shown in FIG. 3 . The nucleotide sequences shown inFIG. 4 are set forth in the sequence listing as VIM unmethylated (SEQ IDNO:18), VIM methylated (SEQ ID NO:19), SEQ ID NO:13 (forward primer),SEQ ID NO:15 (flap probe), and SEQ ID NO: 14 (reverse primer).

FIGS. 5 to 7 each provide data that is described in greater detail inthe Examples section of this application.

DEFINITIONS

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in liquid form,containing one or more analytes of interest.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the like.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally-occurring nucleotides include guanine,cytosine, adenine and thymine (G, C, A and T, respectively).

The term “nucleic acid sample,” as used herein denotes a samplecontaining nucleic acid.

The term “target polynucleotide,” as used herein, refers to apolynucleotide of interest under study. In certain embodiments, a targetpolynucleotide contains one or more target sites that are of interestunder study.

The term “oligonucleotide” as used herein denotes a single strandedmultimer of nucleotides of about 2 to 200 nucleotides. Oligonucleotidesmay be synthetic or may be made enzymatically, and, in some embodiments,are 10 to 50 nucleotides in length. Oligonucleotides may containribonucleotide monomers (i.e., may be oligoribonucleotides) ordeoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 11 to30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to150 or 150 to 200 nucleotides in length, for example.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The term “primer” as used herein refers to an oligonucleotide that has anucleotide sequence that is complementary to a region of a targetpolynucleotide. A primer binds to the complementary region and isextended, using the target nucleic acid as the template, under primerextension conditions. A primer may be in the range of about 15 to about50 nucleotides although primers outside of this length may be used. Aprimer can be extended from its 3′ end by the action of a polymerase. Anoligonucleotide that cannot be extended from its 3′ end by the action ofa polymerase is not a primer.

The term “extending” as used herein refers to any addition of one ormore nucleotides to the 3′ end of a nucleic acid, e.g. by ligation of anoligonucleotide or by using a polymerase.

The term “amplifying” as used herein refers to generating one or morecopies of a target nucleic acid, using the target nucleic acid as atemplate.

The term “denaturing,” as used herein, refers to the separation of anucleic acid duplex into two single strands.

The terms “determining”, “measuring”, “evaluating”, “assessing,”“assaying,” ‘detecting,” and “analyzing” are used interchangeably hereinto refer to any form of measurement, and include determining if anelement is present or not. These terms include both quantitative and/orqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

The term “using” has its conventional meaning, and, as such, meansemploying, e.g., putting into service, a method or composition to attainan end.

As used herein, the term “T_(m)” refers to the melting temperature of anoligonucleotide duplex at which half of the duplexes remain hybridizedand half of the duplexes dissociate into single strands. The T_(m) of anoligonucleotide duplex may be experimentally determined or predictedusing the following formula T_(m)=81.5+16.6(log₁₀[Na⁺])+0.41 (fractionG+C)−(60/N), where N is the chain length and [Na⁺] is less than 1 M. SeeSambrook and Russell (2001; Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., ch. 10).Other formulas for predicting T_(m) of oligonucleotide duplexes existand one formula may be more or less appropriate for a given condition orset of conditions.

As used herein, the term “T_(m)-matched” refers to a plurality ofnucleic acid duplexes having T_(m)s that are within a defined range,e.g., within 5° C. or 10° C. of each other.

As used herein, the term “reaction mixture” refers to a mixture ofreagents that are capable of reacting together to produce a product inappropriate external conditions over a period of time. A reactionmixture may contain PCR reagents and flap cleavage reagents, forexample, the recipes for which are independently known in the art.

The term “mixture”, as used herein, refers to a combination of elements,that are interspersed and not in any particular order. A mixture isheterogeneous and not spatially separable into its differentconstituents. Examples of mixtures of elements include a number ofdifferent elements that are dissolved in the same aqueous solution, or anumber of different elements attached to a solid support at random or inno particular order in which the different elements are not spatiallydistinct. A mixture is not addressable. To illustrate by example, anarray of spatially separated surface-bound polynucleotides, as iscommonly known in the art, is not a mixture of surface-boundpolynucleotides because the species of surface-bound polynucleotides arespatially distinct and the array is addressable.

As used herein, the term “PCR reagents” refers to all reagents that arerequired for performing a polymerase chain reaction (PCR) on a template.As is known in the art, PCR reagents essentially include a first primer,a second primer, a thermostable polymerase, and nucleotides. Dependingon the polymerase used, ions (e.g., Mg 2±) may also be present. PCRreagents may optionally contain a template from which a target sequencecan be amplified.

As used herein, the term “flap assay” refers to an assay in which a flapoligonucleotide is cleaved in an overlap-dependent manner by a flapendonuclease to release a flap that is then detected. The principles offlap assays are well known and described in, e.g., Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447). Forthe sake of clarity, certain reagents that are employed in a flap assayare described below. The principles of a flap assay are illustrated inFIG. 1 . In the flap assay shown in FIG. 1 , an invasive oligonucleotide2 and flap oligonucleotide 4 are hybridized to target 6 to produce afirst complex 8 that contains a nucleotide overlap at position 10. Firstcomplex 8 is a substrate for flap endonuclease. Flap endonuclease 12cleaves flap oligonucleotide 4 to release a flap 14 that hybridizes withFRET cassette 16 that contains a quencher “Q” and a nearby quenchedflourophore “R” that is quenched by the quencher Q. Hybridization offlap 14 to FRET cassette 16 results in a second complex 18 that containsa nucleotide overlap at position 20. The second complex is also asubstrate for flap endonuclease. Cleavage of FRET cassette 16 by flapendonuclease 12 results in release of the fluorophore 22, which producesa fluorescent signal. These components are described in greater detailbelow.

As used herein, the term “invasive oligonucleotide” refers to anoligonucleotide that is complementary to a region in a target nucleicacid. The 3’ terminal nucleotide of the invasive oligonucleotide may ormay not base pair a nucleotide in the target (e.g., which may be5-methylcytosine or uracil, for example).

As used herein, the term “flap oligonucleotide” refers to anoligonucleotide that contains a flap region and a region that iscomplementary to a region in the target nucleic acid. The targetcomplementary regions on the invasive oligonucleotide and the flapoligonucleotide overlap by a single nucleotide such that, when they areannealed to the target nucleic acid, the complementary sequencesoverlap. As is known, if: a) the 3′ terminal nucleotide of the invasivenucleotide and b) the nucleotide that overlaps with that nucleotide inthe flap oligonucleotide both base pair with a nucleotide in the targetnucleic acid, then a particular structure is formed. This structure is asubstrate for an enzyme, defined below as a flap endonuclease, thatcleaves the flap from the target complementary region of the flapoligonucleotide. If the 3′ terminal nucleotide of the invasiveoligonucleotide does not base pair with a nucleotide in the targetnucleic acid, or if the overlap nucleotide in the flap oligononucleotidedoes not base pair with a nucleotide in the target nucleic acid, thecomplex is not a substrate for the enzyme.

The term “flap endonuclease” or “FEN” for short, as used herein, refersto a class of nucleolytic enzymes that act as structure specificendonucleases on DNA structures with a duplex containing a singlestranded 5′ overhang, or flap, on one of the strands that is displacedby another strand of nucleic acid, i.e., such that there are overlappingnucleotides at the junction between the single and double-stranded DNA.FENs catalyze hydrolytic cleavage of the phosphodiester bond at thejunction of single and double stranded DNA, releasing the overhang, orthe flap. Flap endonucleases are reviewed by Ceska and Savers (TrendsBiochem. Sci. 1998 23:331-336) and Liu et al (Annu. Rev. Biochem. 200473: 589-615). FENs may be individual enzymes, multi-subunit enzymes, ormay exist as an activity of another enzyme or protein complex, e.g., aDNA polymerase. A flap endonuclease may be thermostable.

As used herein, the term “cleaved flap” refers to a single-strandedoligonucleotide that is a cleavage product of a flap assay.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleaved flapwith a FRET cassette produces a secondary substrate for the flapendonuclease. Once this substrate is formed, the 5′fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal.

As used herein, the term “flap assay reagents” refers to all reagentsthat are required for performing a flap assay on a substrate. As isknown in the art, flap assays include an invasive oligonucleotide, aflap oligonucleotide, a flap endonuclease and a FRET cassette, asdescribed above. Flap assay reagents may optionally contain a target towhich the invasive oligonucleotide and flap oligonucleotide bind.

As used herein, the term “genomic locus” refers to a defined region in agenome. A genomic locus exists at the same location in the genomes ofdifferent cells from the same individual, or in different individuals. Agenomic locus in one cell or individual has a nucleotide sequence thatis identical or very similar (i.e., more than 99% identical) to the samegenomic locus in a different cell or individual. The difference innucleotide sequence between the same locus in different cells orindividuals may be due to one or more nucleotide substitutions. Agenomic locus may be defined by genomic coordinates, by name, or using asymbol. A genomic locus in a nucleic acid sample that has been treatedwith an agent that modifies unmethylated cytosine to uracil has the samesequence as the genomic locus in an unmethylated sample, except thatunmethylated cytosines in the sequence (but not methylated cytosines)are modified to be become uracils. In amplified copies of a genomiclocus in a nucleic acid sample that has been treated with such an agent,the uracil is converted to thymine.

As used herein, the term “methylation state” refers to the presence orabsence of a methyl group on a cytosine residue at a site ofmethylation. For clarity, a cytosine that is unmethylated will bereferred to as “unmethylated cytosine” or “unmethylated C”, and acytosine that is methylated (i.e., 5-methylcytosine) will be referred toas “methylated cytosine,′”′methylated C,” or “methyl C.”

As used herein, a “site of methylation” refers to the position of acytosine nucleotide that is known to be at least sometimes methylated ina genomic locus. The cytosine at a site of methylation can be anunmethylated cytosine or a methylated cytosine. In other words, the term“site of methylation” refers to a specific cytosine in a genomic locus,the methylation state of which is sought to be determined. The site ofmethylation may be defined by genomic coordinates, or coordinatesrelative to the start codon of a gene, for example.

As will be described in greater detail below, certain embodiments of thesubject method involve treating a nucleic acid sample with an agent thatspecifically converts unmethylated cytosine to uracil by deamination.Therefore, in an untreated sample, the site of methylation will occupiedby an unmethylated cytosine or a methylated cytosine, depending on themethylation status of that site. Likewise, the site of methylation in atreated sample will be occupied by a methylated cytosine or a uracil,depending on the methylation status of that site in the sample prior totreatment.

The term “corresponds to” and grammatical equivalents, e.g.,“corresponding”, as used herein refers to a specific relationshipbetween the elements to which the term refers. For example, anoligonucleotide that corresponds to a sequence in a longer nucleic acidcontains the same nucleotide sequence as or is complementary to anucleotide sequence in the nucleic acid.

In the context of a nucleotide in an oligonucleotide that corresponds toa site of methylation or a nucleotide in an oligonucleotide thatcorresponds to a methylated cytosine, the term “corresponds to” andgrammatical equivalents thereof are intended to identify the nucleotidethat is correspondingly positioned relative to (i.e., positioned acrossfrom) a site of methylation when the two nucleic acids (e.g., anoligonucleotide and genomic DNA containing a methylated cytosine) arealigned or base paired. Again, unless otherwise indicated (e.g., in thecase of a nucleotide that “does not base pair” or “base pairs” with aparticular residue) a nucleotide that “corresponds to” a site ofmethylation base pairs with either a methylated site or an unmethylatedsite. For clarity, in an oligonucleotide, a G or C nucleotide at aposition that corresponds to a methylated cytosine in a sequence, e.g.,a genomic locus, can: a) base pair with a methylated cytosine in thesequence, b) base pair a cytosine that positionally corresponds to themethylated cytosine in an amplified version of the sequence, or c) basepair with a G residue that is complementary to such a cytosine in anamplified sequence.

As will be described in greater detail below, the subject method mayalso involve amplifying a nucleic acid product sample that has beentreated with an agent that specifically converts unmethylated cytosineto uracil (see, for example, Frommer et a. Proc. Natl. Acad. Sci. 199289:1827-1831). As a result of the amplification step, methylatedcytosines are converted to cytosines, and uracils are converted tothymines. The methylation state of a cytosine nucleotide in the initialsample can therefore be evaluated by determining whether a base-pair inthe amplification product that is at the same position as the cytosinein question is a C/G base pair (which indicates that the cytosine inquestion is methylated) or an A/T base pair (which indicates that thecytosine residue is unmethylated). Thus, the methylation status of acytosine in an initial sample can be determined by amplifying a doublestranded product from a sample that has been treated with an agent thatspecifically converts unmethylated cytosine to uracil, and thenexamining the position corresponding to the target cytosine in either ofthe strands (i.e., either the top strand or the bottom strand) of theamplification product to determine whether an A or T is present (whichindicates that the cytosine in question is methylated), or if a G or Cis present (which indicates that the cytosine in question ismethylated). Thus, in the context of an oligonucleotide that hybridizesto a double stranded amplification product produced by amplification ofa genomic locus from a sample that has been treated with an agent thatspecifically converts unmethylated cytosine to uracil, a nucleotide that“corresponds to” a site of methylation is a nucleotide that base pairswith either the top strand or the bottom strand at the site ofmethylation.

As used herein, a “sequence that is methylated” is a nucleotide sequencethat contains a site of methylation, i.e., a cytosine nucleotide that isknown to be at least sometimes methylated.

As used herein, the term “unmethylated”, with reference a nucleotidesequence, refers to the copies of a sequence that are not methylated.

As used herein, the term “methylated”, with reference a nucleotidesequence, refers to copies of a sequence that contain 5-methylcytosine.Methylation of a genomic locus may, e.g., alter the expression of aprotein, which causes a phenotypic change (e.g., a cancer-relatedphenotype) in the cells that have such a methylated locus.Alternatively, methylation of a genomic locus may be silent.

A sample that comprises “both unmethylated and methylated copies of agenomic locus” and grammatical equivalents thereof, refers to a samplethat contains multiple DNA molecules of the same genomic locus, wherethe sample contains both unmethylated copies of the genomic locus andmethylated copies of the same locus. In this context, the term “copies”is not intended to mean that the sequences were copied from one another.Rather, the term “copies” in intended to indicate that the sequences areof the same locus in different cells or individuals. In other words, asample contains a mixture of nucleic acid molecules having the samenucleotide sequence, except that some of the molecules containmethylated cytosine residues.

As used herein, the term “degree of methylation” refers to the relativenumber, percentage, or fraction of members of a particular targetnucleotide species within a sample that are methylated compared to thosemembers of that particular target nucleotide species that are notmethylated.

As used herein, the term “an agent that modifies unmethylated cytosineto uracil” refers to any agent that specifically deaminates unmethlyatedcytosine to produce uracil. Such agents are specific in that they do notdeaminate 5-methylcytosine to produce uracil. Bisulfite is an example ofsuch an agent.

As used herein, the term “a treated nucleic acid sample” is a nucleicacid sample that has been treated with an agent that modifiesunmethylated cytosine to uracil.

As used herein, the term “initial sample” refers to a sample that hasnot been treated with an agent that modifies unmethylated cytosine touracil

As used herein the term “nucleotide sequence” refers to a contiguoussequence of nucleotides in a nucleic acid. As would be readily apparent,the number of nucleotides in a nucleotide sequence may vary greatly. Inparticular embodiments, a nucleotide sequence (e.g., of anoligonucleotide) may be of a length that is sufficient for hybridizationto a complementary nucleotide sequence in another nucleic acid. In theseembodiments, a nucleotide sequence may be in the range of at least 10 to50 nucleotides, e.g., 12 to 20 nucleotides in length, although lengthsoutside of these ranges may be employed in many circumstances.

As used herein the term “fully complementary to” in the context of afirst nucleic acid that is fully complementary to a second nucleic acidrefers to a case when every nucleotide of a contiguous sequence ofnucleotides in a first nucleic acid base pairs with a complementarynucleotide in a second nucleic acid.

As used herein the term a “primer pair” is used to refer to two primersthat can be employed in a polymerase chain reaction to amplify a genomiclocus. A primer pair may in certain circumstances be referred to ascontaining “a first primer” and “a second primer” or “a forward primer”and “a reverse primer”. Use of any of these terms is arbitrary and isnot intended to indicate whether a primer hybridizes to a top strand orbottom strand of a double stranded nucleic acid.

A “CpG” island is defined as a region of DNA of greater than 500 bp witha G/C content of at least 55% and an observed CpG/expected CpG ratio ofat least 0.65, as defined by Takai et al Proc. Natl. Acad. Sci. 2002 99:3740-3745). Use of this formula to identify CpG islands excludes otherGC-rich genomic sequences such as Alu repeats.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In the following description, the skilled artisan will understand thatany of a number of polymerases and flap endonucleases could be used inthe methods, including without limitation, those isolated fromthermostable or hyperthermostable prokaryotic, eukaryotic, or archaealorganisms. The skilled artisan will also understand that the enzymesthat are used in the method, e.g., polymerase and flap endonuclease,include not only naturally occurring enzymes, but also recombinantenzymes that include enzymatically active fragments, cleavage products,mutants, and variants of wild type enzymes.

In further describing the method, the reagent mixture used in the methodwill be described first, followed by a description of the method bywhich a sample may be treated and the reaction conditions that may beused in the method.

Reaction Mixture

The reaction mixture may vary depending how the reaction is performed,e.g., whether, for example, one or both of the first and second primershybridize to methylated sequences, or whether the first primer (which isused for amplification of a genomic locus) is also employed as aninvasive oligonucleotide in the flap assay, in which case no distinctinvasive oligonucleotide need be included in the assay mixture.

In general terms, the reaction mixture used in the method may generallycontain: a) amplification reagents comprising a thermostable polymerase,nucleotides, a first primer and a second primer for amplifying a targetgenomic locus from a treated nucleic acid sample; wherein: i. the firstprimer hybridizes to a methylated sequence in the genomic locus andoptionally contains a 3′ G or C terminal nucleotide that corresponds toa methylated cytosine in the genomic locus; and ii. the reagentspreferentially amplify methylated copies of the genomic locus, toproduce an amplified sample; b) flap assay reagents comprising a flapendonuclease, a FRET cassette, a flap oligonucleotide and, if the firstprimer does not contain a 3′ terminal nucleotide that corresponds to themethylated cytosine, an invasive oligonucleotide, that is distinct fromthe first primer, that has a 3′ terminal G or C nucleotide thatcorresponds to the methylated cytosine; and c) the treated nucleic acidsample, wherein the treated nucleic acid sample is made by treating aninitial nucleic acid sample comprising both methylated copies andunmethylated copies of the genomic locus with an agent that modifiesunmethylated cytosine to uracil. The flap oligonucleotide contains a Gor C nucleotide at a position that corresponds to the methylatedcytosine. The reaction mixture is characterized in that it can amplifyand detect the presence of methylated copies of the genomic locus in thesample.

As noted above, the amplification generally employs a first primer thathybridizes to a methylated sequence in a genomic locus andpreferentially amplifies methylated copies of the genomic locus. Incertain embodiments, the first primer may contain one or morenucleotides (e.g., G residues) that base pair with correspondingmethylated cytosine nucleotides in the methylated sequence (which wouldhave been converted to a uracil if they were unmethylated). Inparticular embodiments, the first primer may contain up to 3 or 4nucleotides that base pair with corresponding methylated cytosines in amethylated sequence, particularly toward the 3′ end of the primerthereby making the primer a methylation specific primer in that itpreferentially amplifies methylated copies of the genomic locus. In oneembodiment, the primer may contain a 3′ terminal nucleotide that basepairs with a methylated cytosine in the methylated sequence, or basepairs with a G residue in an amplicon complementary to a methylatedcytosine, as well as 1, 2 or 3 further nucleotides that base pair withother methylated cytosines or their complements in the sequence. Thus,the first primer may contain one or more internal G or C nucleotides atpositions that correspond to a corresponding number of second methylatedcytosine in the genomic locus.

While thereby preferential amplification of methylated copies of agenomic locus may be done using a pair of primers in which only one ofthe primers is methylation specific, in particular embodiments, both thefirst and second primers may be methylation-specific in that they bothhybridize to methylated sequences in the genomic locus.

The design of the methylation-specific primers that may be present inthe reaction mixture may be adapted from, e.g., the primer designmethods described by, e.g., Herman et al (Methylation-specific PCR: anovel PCR assay for methylation status of CpG islands. Proc. Natl. Acad.Sci. 1996 93: 9821-6) and Ehrich et al (Quantitative high-throughputanalysis of DNA methylation patterns by base-specific cleavage and massspectrometry. Proc. Natl. Acad. Sci. 2005 102: 15785-90), as well asthose reviewed in Li (Designing PCR primer for DNA methylation mapping.Methods Mol Biol. 2007 402: 371-84), Derks et al (Methylation-specificPCR unraveled. Cell Oncol. 2004 26:291-9) and Cottrell et al (Sensitivedetection of DNA methylation. Ann N Y Acad. Sci. 2003 983:120-30). Sincethe identities of many if not most CpG islands in the human and othergenomes are known, (see, e.g., Lauss et al Br. J. Cancer.MethCancerDB—aberrant DNA methylation in human cancer 2008 98: 816-817;Wang et al Bioinformatics, An evaluation of new criteria for CpG islandsin the human genome as gene markers 2003 20: 1170-1177) the design ofmethylation-specific primers for analysis of a number of differentgenomic loci may be done without undue effort.

As noted above, the presence of distinct invasive oligonucleotides inthe reaction mixture may depend on whether the first primer is alsoemployed as an invasive oligonucleotide in the flap assay. As such, insome embodiments, the first primer contains a 3′ terminal nucleotidethat base pairs with a methylated cytosine in the sequence to which theprimer binds. In these embodiments, the first primer may be employed asan invasive oligonucleotide in the flap assay and, as such, the reactionmixture need not contain an invasive oligonucleotide in addition to thefirst primer. In other embodiments, the first primer does not contain a3′ terminal nucleotide that base pairs with a methylated cytosine in themethylated sequence. In these embodiments, the reaction mixture maycontain a distinct invasive oligonucleotide that contains a 3′ terminalG or C nucleotide that corresponds to the site of methylation. In theseembodiments, the 3′ terminal nucleotide of the invasive oligonucleotidemay base pair with a site of methylation that is internal to thesequences of the primers in the amplification product.

In alternative embodiments, the reaction mixture may comprise a firstprimer that contains a 3′ terminal nucleotide that base pairs with amethylated cytosine or its complement in the genomic locus as well as aninvasive oligonucleotide that contains a 3′ terminal G or C nucleotidethat corresponds to the site of methylation. In these embodiments, the3′ terminal nucleotide of the first primer and the 3′ terminalnucleotide of the invasive oligonucleotide base pair with nucleotides atdifferent sites of methylation in the genomic locus. In one embodiment,the 3′ terminal nucleotide of the invasive oligonucleotide base pairswith a site of methylation that is internal to the sequences of theprimers in the amplification product.

In particular embodiments, a separate invasive oligonucleotide maycontain other nucleotides (e.g., G or C nucleotides) in addition to the3′ terminal nucleotide that base pair with nucleotides at other sites ofmethylation. In other words, a separate invasive oligonucleotides maycontain one or more (e.g., 1, 2, 3 or 4 or more) internal G or Cnucleotides that correspond to methylated cytosines. These internalnucleotides increase the specificity of binding of the invasiveoligonucleotide to nucleic acid that has been amplified from methylatedcopies of the genomic locus, thereby increasing the fidelity ofdetection. In a similar manner, the portion of the flap oligonucleotidethat hybridizes to the amplification product may contain one or more(e.g., 1, 2, 3 or 4 or more) internal G or C nucleotides that correspondto methylated cytosines, which serve to increase the specificity ofbinding of the flap oligonucleotide to nucleic acid that has beenamplified from methylated copies of the genomic locus, therebyincreasing the fidelity of detection. Thus, in some embodiments, theflap oligonucleotide may contain one or more internal G or C nucleotidepositions that corresponds to a corresponding number of secondmethylated cytosines in the genomic locus.

The exact identities and concentrations of the reagents present in thereaction mixture may be similar to or the same as those independentlyemployed in PCR and flap cleavage assays, with the exception that thereaction mixture contains Mg²⁺ at a concentration that is higher thanemployed in conventional PCR reaction mixtures (which contain Mg²⁺ at aconcentration of between about 1.8 mM and 3 mM). In certain embodiments,the reaction mixture described herein contains Mg²⁺ at a concentrationof 4 mM to 10 mM, e.g., 6 mM to 9 mM. Exemplary reaction buffers and DNApolymerases that may be employed in the subject reaction mixture includethose described in various publications (e.g., Ausubel, et al., ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 ColdSpring Harbor, N.Y.). Reaction buffers and DNA polymerases suitable forPCR may be purchased from a variety of suppliers, e.g., Invitrogen(Carlsbad, CA), Qiagen (Valencia, CA) and Stratagene (La Jolla, CA).Exemplary polymerases include Taq, Pfu, Pwo, UlTma and Vent, althoughmany other polymerases may be employed in certain embodiments. Guidancefor the reaction components suitable for use with a polymerase as wellas suitable conditions for their use is found in the literature suppliedwith the polymerase. Primer design is described in a variety ofpublications, e.g., Diffenbach and Dveksler (PCR Primer, A LaboratoryManual, Cold Spring Harbor Press 1995); R. Rapley, (The Nucleic AcidProtocols Handbook (2000), Humana Press, Totowa, N.J.); Schena and Kwoket al., Nucl. Acid Res. 1990 18:999-1005). Primer and probe designsoftware programs are also commercially available, including withoutlimitation, Primer Detective (ClonTech, Palo Alto, Calif.), Lasergene,(DNASTAR, Inc., Madison, Wis.), Oligo software (National Biosciences,Inc., Plymouth, Minn), and iOligo (Caesar Software, Portsmouth, N.H).

Exemplary flap cleavage assay reagents are found in Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447).Appropriate conditions for flap endonuclease reactions are either knownor can be readily determined using methods known in the art (see, e.g.,Kaiser et al., J. Biol. Chem. 274:21387-94, 1999). Exemplary flapendonucleases that may be used in the method include, withoutlimitation, Thermus aquaticus DNA polymerase I, Thermus thermophilus DNApolymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1,Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1,Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1,CLEAVASE™ (Third Wave, Inc., Madison, Wis.), S. cerevisiae RTH1, S.cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′exonuclease, Pyroccus horikoshii FEN-1, human exonuclease 1, calf thymus5′-3′ exonuclease, including homologs thereof in eubacteria, eukaryotes,and archaea, such as members of the class II family ofstructure-specific enzymes, as well as enzymatically active mutants orvariants thereof. Descriptions of cleaving enzymes can be found in,among other places, Lyamichev et al., Science 260:778-83, 1993; Eis etal., Nat. Biotechnol. 19:673-76, 2001; Shen et al., Trends in Bio. Sci.23:171-73, 1998; Kaiser et al. J. Biol. Chem. 274:21387-94, 1999; Ma etal., J. Biol. Chem. 275:24693-700, 2000; Allawi et al., J. Mol. Biol.328:537-54, 2003; Sharma et al., J. Biol. Chem. 278:23487-96, 2003; andFeng et al., Nat. Struct. Mol. Biol. 11:450-56, 2004.

In particular embodiments, the reaction mix may contain reagents forassaying multiple (e.g., at least 2, 3, 4 or more) different targetssequences in parallel. In these cases, the reaction mix may containmultiple pairs of PCR primers, multiple different flap oligonucleotideshaving different flaps, and multiple different FRET cassettes fordetecting the different flaps, once they are cleaved. In one embodiment,oligonucleotides in a mixture may have common flaps but differentbinding sequences to allow for, for example, any of a number ofmethylated cytosines to cleave a common FRET cassette and report asignal where a single fluorophore is indicative of the presence of amethylated cytosine. In this embodiment, which site is methylated in thesample may be determined after the presence of a methylated cytosine hasidentified. Optionally, the reaction may contain multiple invasiveoligonucleotides if one of the PCR primers is not used as an invasiveoligonucleotide. Upon cleavage of the FRET cassettes, multipledistinguishable fluorescent signals may be observed. The fluorophore maybe selected from, e.g., 6-carboxyfluorescein (FAM), which has excitationand emission wavelengths of 485 nm and 520 nm respectively, Redmond Red,which has excitation and emission wavelengths of 578 nm and 650 nmrespectively and Yakima Yellow, which has excitation and emissionwavelengths of 532 nm and 569 nm respectively, and Quasor670 which hasexcitation and emission wavelengths of 644 nm and 670 nm respectively,although many others could be employed. In certain cases, at least oneof the PCR primer pairs, flap oligonucleotides and FRET cassettes may befor the detection of an internal control. In such an assay, the controlreagents may be, e.g., for amplification and detection of a locus thatis not methylated or, for example, or for the amplification anddetection of copies of the same locus. In these embodiments a reactionmixture may contain, in addition to other necessary reagents, at leastan oligonucleotide having a 3′ terminal nucleotide that base pairs withan A or T residue at a site of methylation, thereby providing for thedetection of unmethylated copies of the genomic locus. These embodimentsmay also employ primers that amplified the unmethylated copies of thegenomic locus.

As would be apparent, the various oligonucleotides used in the methodare designed so as to not interfere with each other. For example, inparticular embodiments, the flap oligonucleotide may be capped at its 3′end, thereby preventing its extension. Likewise, in certain embodimentsthe invasive oligonucleotide may also be capped at its 3′ end if it notused as one of the PCR primers. In particular embodiment, if theinvasive oligonucleotide is not used as one of the PCR primers, then theinvasive oligonucleotide may be present at a concentration that is inthe range of 5% to 50%, e.g., 10% to 40% of the concentration of the PCRprimers. Further, in certain cases, the T_(m)s of the flap portion andthe target complementary regions of the flap oligonucleotide mayindependently be at least 10° C. lower (e.g., 10-20° C. lower) than theT_(m)s of the PCR primers, which results in: a) less hybridization ofthe flap oligonucleotide to the target nucleic acid at highertemperatures (65° C. to 75° C.) and b) less hybridization of any cleavedflap to the FRET cassette at higher temperatures (65° C. to 75° C.),thereby allowing the genomic locus to be amplified by PCR at atemperature at which the flap does not efficiently hybridize.

In a multiplex reaction, the primers may be designed to have similarthermodynamic properties, e.g., similar T_(m)s, G/C content, hairpinstability, and in certain embodiments may all be of a similar length,e.g., from 18 to 30 nt, e.g., 20 to 25 nt in length. The other reagentsused in the reaction mixture may also be T_(m) matched.

The assay mixture may be present in a vessel, including withoutlimitation, a tube; a multi-well plate, such as a 96-well, a 384-well, a1536-well plate; and a microfluidic device. In certain embodiments,multiple multiplex reactions are performed in the same reaction vessel.Depending on how the reaction is performed, the reaction mixture may beof a volume of 5 μl to 200 e.g., 10 μl to 100 although volumes outsideof this range are envisioned.

In certain embodiments, a subject reaction mix may further contain anucleic acid sample. In particular embodiments, the sample may containgenomic DNA or an amplified version thereof (e.g., genomic DNA amplifiedusing the methods of Lage et al, Genome Res. 2003 13: 294-307 orpublished patent application US20040241658, for example). In exemplaryembodiments, the genomic sample may contain genomic DNA from a mammaliancell, such as, a human, mouse, rat, or monkey cell. The sample may bemade from cultured cells or cells of a clinical sample, e.g., a tissuebiopsy, scrape or lavage or cells of a forensic sample (i.e., cells of asample collected at a crime scene). In particular embodiments, thegenomic sample may be from a formalin fixed paraffin embedded (FFPE)sample.

Method for Sample Analysis

In particular embodiments, the nucleic acid sample may be obtained froma biological sample such as cells, tissues, bodily fluids, and stool.Bodily fluids of interest include but are not limited to, blood, serum,plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid,tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovialfluid, urine, amniotic fluid, and semen. In particular embodiments, asample may be obtained from a subject, e.g., a human, and it may beprocessed prior to use in the subject assay. For example, the nucleicacid may be extracted from the sample prior to use, methods for whichare known. For example, DNA can be extracted from stool from any numberof different methods, including those described in, e.g, Coll et al (J.of Clinical Microbiology 1989 27: 2245-2248), Sidransky et al (Science1992 256: 102-105), Villa (Gastroenterology 1996 110: 1346-1353) andNollau (BioTechniques 1996 20: 784-788), and U.S. Pat. Nos. 5,463,782,7,005,266, 6,303,304 and 5,741,650. Commercial DNA extraction kits forthe extraction of DNA from stool include the QTAarrip stool mini kit(QTAGEN, Hilden, Germany), Instagene Matrix (Bio-Rad, Hercules, Calif.),and RapidPrep Micro Genomic DNA isolation kit (Pharmacia Biotech Inc.,Piscataway, N.J.), among others.

Treatment of an initial nucleic acid sample to produce a treated nucleicacid sample involves contacting the initial nucleic acid sample with anagent that modifies unmethylated cytosine to uracil under conditions(e.g., a length of time and temperature, etc.) for the unmethylatedcytosines in the nucleic acid to deaminated, thereby converting intouracils. Such methods are known and are described in, e.g., Clark et al(Nucleic Acids Res. 1994 22:2990-7), McDonald et al (Biotechniques. 199722: 272-4), Herman et al (Proc. Natl. Acad. Sci. 1996 93:9821-6) andPaul et al (Biotechniques 1996 21:126-33) as well as a variety of otherreferences.

After treatment, the sample, referred to herein the “treated sample”, iscombined with other reagents to produce the reaction mixture describedabove, and then subjected to one or more sets of thermocyclingconditions.

Exemplary conditions include, for example those described in Allawi etal (J Clin Microbiol. 2006 44: 3443-3447). In one embodiment, thereaction mixture may be subjected to conventional PCR thermocycling(i.e., multiple rounds of denaturation at a temperature of over 90° C.,e.g., at about 95° C., annealing at a temperature of 65° C. to 75° C.and extension at a temperature of 65° C. to 75° C.) followed by a periodat high temperature to denature the thermostable polymerase (e.g., about99° C.), and then a period at a temperature that is about ° C. below theextension temperature during which fluorescence is detected.

In other embodiments, the reaction mixture may be subject to cyclingconditions in which an increase in the amount of amplified product(indicated by the amount of fluorescence) can be measured in real-time,where the term “real-time” is intended to refer to a measurement that istaken as the reaction progresses and products accumulate. Themeasurement may be expressed as an absolute number of copies or arelative amount when normalized to a control nucleic acid in the sample.In one real time embodiment, the reaction may be subjected to thethermocycling conditions described in, e.g., Tadokoro (J. Vir. Methods2009 155: 182-186). In this embodiment, the reaction mixture may besubjected to multiple cycles of four steps that include a denaturationstep at a temperature of over 90° C., e.g., at about 95° C., annealingat a temperature in the range of 61° C. to 69° C., flap cleavage at atemperature of 50° C., and extension at a temperature of 72° C. In thisembodiment, fluorescence can be monitored in each cycle to provide areal time measurement of the amount of product that is accumulating inthe reaction mixture.

In an alternative embodiment, the reaction mixture may be subjected tothe following thermocycling conditions: a first set of 5 to 15 (e.g., 8to 12) cycles of: i. a first temperature of at least 90° C.; ii. asecond temperature in the range of 60° C. to 75° C. (e.g., 65° C. to 75°C.); iii. a third temperature in the range of 65° C. to 75° C.; followedby: a second set of 20-50 cycles of: i. a fourth temperature of at least90° C.; ii. a fifth temperature that is at least 10° C. lower than thesecond temperature (e.g., in the range of 50° C. to 55° C.); and iii. asixth temperature in the range of 65° C. to 75° C. No additionalreagents need to be added to the reaction mixture during thethermocycling, e.g., between the first and second sets of cycles. Inparticular embodiments, the thermostable polymerase is not inactivatedbetween the first and second sets of conditions, thereby allowing thetarget to be amplified during each cycle of the second set of cycles. Inparticular embodiments, the second and third temperatures are the sametemperature such that “two step” thermocycling conditions are performed.Each of the cycles may be independently of a duration in the range of 10seconds to 3 minutes, although durations outside of this range arereadily employed. In each cycle of the second set of cycles (e.g., whilethe reaction is in the fifth temperature), a signal generated bycleavage of the flap probe may be measured to provide a real-timemeasurement of the amount of product in the sample.

Some of the principles of an example of the subject method areschematically illustrated in FIG. 2 . As noted above, however, themethod may be performed in many different ways, e.g., by employing thefirst primer as an invasive oligonucleotide or by using a singlemethylation specific primer. As such, FIG. 2 shows an example of themethod and should not be used to limit to only the embodiment shown.

With reference to FIG. 2 , the method includes treating an initialsample 30 that comprises both methylated copies of a genomic locus 32and unmethylated copies of the genomic locus 34 with an agent thatmodified unmethylated cytosine to uracil to produce treated sample 36.This treatment converts unmethylated cytosines, e.g., 38, to uracilse.g., Methylated cytosines, e.g., 42 remain as methylated cytosinesduring the treatment. Treated sample 36 is then combined with the otherreagents.

Product 44 is then amplified from treated sample 36 using first primer46 and second primer 48, where the first primer hybridizes to amethylated sequence in the locus and the amplifying preferentiallyamplifies methylated copies of the genomic locus, to produce anamplified sample 50. As illustrated, both first primer 46 and the secondprimer 48 hybridize to methylated sequences. However, in practice, onlyone of the primers need hybridize to a methylated sequence. Inparticular embodiments and as noted above, the first primer may have a3′ terminal nucleotide that base pairs with a methylated cytosine,although this is not necessary if the reaction employs an invasiveoligonucleotide that is distinct from the first primer. Such primersgenerally contain G nucleotides at sites of methylation, therebyallowing the primers to hybridize and extend more efficiently fromsequences that contain methylated cytosine (which are not affected bythe treatment) as opposed to sequences that contain unmethylatedcytosine (which are converted to U's by the treatment). As illustratedin FIG. 2 , the presence of product 44 in amplified sample 50 may bedetected using a flap assay that employs invasive oligonucleotide 52that has a 3′ terminal nucleotide that base pairs with a G or C residuethat corresponds to a site of methylation. The choice of the G or Cresidue is determined by whether the nucleotide that corresponds to thesite of methylation to be detected is in the top or bottom strand of theamplification product. As shown, invasive oligonucleotide 52 has aterminal G nucleotide because it base pairs with a C corresponding to asite of methylation in initial sample 30. Again, as noted above, theembodiments illustrated in FIG. 2 employs a separate invasiveoligonucleotide. In other embodiments, the first oligonucleotide may beemployed as an invasive oligonucleotide in the method and, as such,there is no need to use a separate invasive oligonucleotide in theassay. As shown in FIG. 2 , the 3′ terminal nucleotide of the invasiveoligonucleotide base pairs with an “internal” site of methylation in thesense that the site is within the amplified region and not part of thefirst and second primers.

As shown, the flap assay relies on the cleavage of complex 53 thatcontains an invasive oligonucleotide 52, flap oligonucleotide 56, andthe bottom strand of product 44 by a flap endonuclease (not shown) torelease flap 60. Released flap 60 then hybridizes to FRET cassette 62 toform a second complex 63 that is cleaved by the flap endonuclease tocleave the fluorophore from the complex and generate fluorescent signal64 that can be measured to indicate the amount of product 44 in theamplified sample.

Certain aspects of the method described above are illustrated by examplein FIGS. 3 and 4 . FIG. 3 show the nucleotide sequences of methylatedand unmethylated copies of a fragment of the human vimentin gene (VIM),before (SEQ ID NOS:1 and 2) and after bisulfite treatment (SEQ ID NOS: 3and 4). FIG. 4 shows the nucleotide sequences of an exemplary forwardprimer, an exemplary reverse primer, and an exemplary flapoligonucleotide, aligned with the bisufite-treated fragment shown inFIG. 3 .

The amount of product in the sample may be normalized relative to theamount of a control nucleic acid present in the sample, therebydetermining a relative amount of the methylated copies of the genomiclocus in the sample. In some embodiments, the control nucleic acid maybe a different locus to the genomic locus and, in certain cases, may bedetected using a flap assay that employs an invasive oligonucleotidehaving a 3′ terminal nucleotide that base pairs with an A or T residueat the same site of methylation, thereby detecting the presence ofunmethlyated copies of the genomic locus. The control may be measured inparallel with measuring the product in the same reaction mixture or adifferent reaction mix. If the control is measured in the same reactionmixture, the flap assay may include further reagents, particularly asecond invasive oligonucleotide, a second flap probe having a secondflap and a second FRET cassette that produces a signal that isdistinguishable from the FRET cassette used to detect the product. Inparticular embodiments, the reaction mixture may further comprise PCRreagents and flap reagents for amplifying and determining themethylation state of another genomic locus that is known to bemethylated in some samples.

In certain cases, fluorescence indicating the amount of cleaved flap canbe detected by an automated fluorometer designed to perform real-timePCR having the following features: a light source for exciting thefluorophore of the FRET cassette, a system for heating and coolingreaction mixtures and a fluorometer for measuring fluorescence by theFRET cassette. This combination of features, allows real-timemeasurement of the cleaved flap, thereby allowing the amount of targetnucleic acid in the sample to be quantified. Automated fluorometers forperforming real-time PCR reactions are known in the art and can beadapted for use in this specific assay, for example, the ICYCLER™ fromBio-Rad Laboratories (Hercules, Calif.), the Mx3000P™, the MX3005P™ andthe MX4000™ from Stratagene (La Jolla, Calif.), the ABI PRISM™ 7300,7500, 7700, and 7900 Taq Man (Applied Biosystems, Foster City, Calif.),the SMARTCYCLER™, ROTORGENE 2000™ (Corbett Research, Sydney, Australia)and the GENE XPERT™ System (Cepheid, Sunnyvale, Calif.) and theLIGHTCYCLER™ (Roche Diagnostics Corp., Indianapolis, Ind.). The speed oframping between the different reaction temperatures is not critical and,in certain embodiments, the default ramping speeds that are preset onthermocyclers may be employed.

In certain cases, the method may further involve graphing the amount ofcleavage that occurs in several cycles, thereby providing a real timeestimate of the abundance of the nucleic acid target. The estimate maybe calculated by determining the threshold cycle (i.e., the cycle atwhich this fluorescence increases above a predetermined threshold; the“Ct” value or “Cp” value). This estimate can be compared to a control(which control may be assayed in the same reaction mix as the genomiclocus of interest) to provide a normalized estimate. The thermocyclermay also contain a software application for determining the thresholdcycle for each of the samples. An exemplary method for determining thethreshold cycle is set forth in, e.g., Luu-The et al (Biotechniques 200538: 287-293).

A device for performing sample analysis is also provided. In certainembodiments, the device comprises: a) a thermocycler programmed toperform the above-described method and b) a vessel comprising theabove-described reaction mixture.

Utility

The method described finds use in a variety of applications, where suchapplications generally include sample analysis applications in which thepresence of a methylated sequence in a given sample is detected.

In some embodiments, a biological sample may be obtained from a patient,and the sample may be analyzed using the method. In particularembodiments, the method may be employed to identify and/or estimate theamount of methylated copies of a genomic locus that are in a biologicalsample that contains both unmethylated copies of a genomic locus andmethylated copies of the genomic locus In this example, the sample maycontain at least 100 times (e.g., at least 1,000 times, at least 5,000times, at least 10,000 times, at least 50,000 times or at least 100,000times) more wild type copies of the genomic locus than mutant copies ofthe genomic locus.

In particular, the above-described methods may be employed to diagnose,to predict a response to treatment, or to investigate a cancerouscondition or another mammalian disease that is associated with aberrantmethylation including but not limited to: a) imprinting disordersincluding Beckwith-Wiedemann syndrome (associated with the BWS locus at11p15.5), Prader-Willi syndrome (associated with an imprinted region at15p11-q13), Angelman syndrome (also associated with an imprinted regionat 15p11-q13), Albright hereditary osteodystrophy (associated with animprinting at GNAS), pseudo-hypoparathyroidism types 1a and 1b(associated with imprinting at HYMAI, PLAG1 and ZAC-AS), transientneonatal diabetes mellitus and certain cancers (associated with theIGF2/H19 locus, the CDKN1C gene, DIRAS3 gene, and the MEST gene); b)repeat instability diseases including fragile X syndrome (associatedwith methylation at FRAXA) and facioscapulohumeral muscular dystrophy(associated with methylation at the FSHD locus); c) diseases caused by adefect in methylation pathways such as systemic lupus erythematosus,immunodeficiency (SLE, which is a result of global hypomethylation of Tcells) and centromeric instability and facial anomalies syndrome (ICF)and d) other diseases such as alpha-thalassemia/mental retardationsyndrome, X-linked (associated with abnormal methylation of ATRX). Thesediseases are reviewed in Robertson (DNA methylation and human diseaseNat. Reviews 2005 6: 597-610). The method described above, may, forexample, be used to identify aberrant methylation in an unborn child.

Hypermethylation of CpG islands in various loci is associated withvarious cancers. Without being bound to any particular theory, it isbelieved that methylation inactivates the expression of genes, includingtumor suppressor genes, cell cycle related genes, DNA mismatch repairgenes, hormone receptors and tissue or cell adhesion molecules. Forexample, tumor-specific deficiency of expression of the DNA repair genesMLH1 and MGMT and the tumor suppressors, p16, CDKN2 and MTS1, has beendirectly correlated to hypermethylation. Increased CpG islandmethylation is thought to result in the inactivation of these genesresulting in increased levels of genetic damage, predisposing cells tolater genetic instability which then contributes to tumor progression.

Hypermethylation has been associated with several cancers, asillustrated in Table 1 (adapted from Das et al J. Clin. Oncol. 200422:4632-42). Thus, the method may be employed as a diagnostic for thosecancers.

TABLE 1 Methylated Putative Role in Tumor Gene Development Site of TumorAPC Deranged regulation of cell Breast, Lung, proliferation, cellmigration, cell Esophageal adhesion, cytoskeletal reorganization, andchromosomal stability BRCA1 Implicated in DNA repair and Breast, Ovariantranscription activation CDKN2A/p16 Cyclin-dependent kinase inhibitorGIT, Head and neck, NHL, Lung DAPKI Calcium/calmodulin-dependent Lungenzyme that phosphorylates serine/threonine residues on proteins;Suppression of apoptosis E-cadherin Increasing proliferation, invasion,Breast, Thyroid, and/or metastasis Gastric ER Hormone resistance Breast,Prostate GSTP1 Loss of detoxification of active Prostate, Breast,metabolites of several carcinogens Renal hMLH1 Defective DNA mismatchrepair Colon, Gastric, and gene mutations Endometrium, Ovarian MGMTp53-related gene involved in DNA Lung, Brain repair and drug resistancep15 Unrestrained entry of cells into Leukemia, Lymphoma, activation andproliferation Squamous cell carcinoma, lung RASSFIA Loss of negativeregulator control Lung, Breast, Ovarian, of cell proliferation throughKidney, inhibition of G₁/S-phase Nasopharyngeal progression Rb Failureto repress the transcription Retinoblastoma, of cellular genes requiredfor DNA Oligodendroglioma replication and cell division VHL Altered RNAstability through and Renal cell cancer erroneous degradation of RNA-bound proteins

Abbreviations: APC, adenomatous polyposis coli; BRCA1, breast cancer 1;CDKN2A/p16, cyclin-dependent kinase 2A; DAPK1, death-associated proteinkinase 1; ER, estrogen receptor; GSTP1, glutathione S-transferase Pi 1;hMLH1, Mut L homologue 1; MGMT, 0-6 methylguanine-DNA methyltransferase;RASSF1A, Ras association domain family member 1; Rb, retinoblastoma;VHL, von Hippel-Lindau; GIT, gastrointestinal tract; NHL, non-Hodgkin'slymphoma.

The hypmermethylation of the following genes is also associated withcancer: PYCARD, CDH13, COX2, DAPK1, ESR1, GATA4, SYK, MLH1, TP73, PRDM2,PGR, SFRP1, SOCS1, SOCS3, STK11, TME1-1-2, THBS1, RASSFS, PRKCDBP, MGMT,CDKN2A, SFRP1, TME1-1-2, HS3ST2 (30ST2), RASSF1A, GATA4 and RARB.

In these embodiments, the method may be employed to detect aberrantmethylation (e.g., hypermethylation or hypomethylation) in a gene, whichaberrant methylation may be associated with, e.g., breast cancer,melanoma, renal cancer, endometrial cancer, ovarian cancer, pancreaticcancer, leukemia, colorectal cancer, prostate cancer, mesothelioma,glioma, medullobastoma, polycythemia, lymphoma, sarcoma or multiplemyeloma, etc.

The use of DNA methylation markers for diagnosing cancers has beenreviewed in a variety of publications such as: Qureshi et al (Int JSurg. 2010 Utility of DNA methylation markers for diagnosing cancer.8:194-8), Muraki et al (Oncol Rep. 2009 Epigenetic DNA hypermethylation:clinical applications in endometrial cancer 22:967-72), Balch et al(Endocrinology. 2009 Minireview: epigenetic changes in ovarian cancer.150:4003-11), Pfeifer (Semin Cancer Biol. 2009 DNA methylation patternsin lung carcinomas 19:181-7), Szalmas et al (Semin Cancer Biol. 2009Epigenetic alterations in cervical carcinogenesis 19:144-52), Hoque(Expert Rev Mol Diagn. 2009 DNA methylation changes in prostate cancer:current developments and future clinical implementation 9:243-57), andCampan et al (Curr Top Microbiol Immunol. 2006 DNA methylation profilesof female steroid hormone-driven human malignancies 310:141-78).

In one embodiment, the method may be employed to detected methylation infecal DNA, thereby providing a diagnostic for colorectal cancer. Inthese embodiments, the method may be employed to investigate methylationof BMP3, EYA2, ALX4, or Vimentin, for example. These genes and theirmethylation are described in, for example, Chen et al (J Natl CancerInst. 2005 Detection in fecal DNA of colon cancer-specific methylationof the nonexpressed vimentin gene. 97:1124-32), Zou et al (CancerEpidemiol Biomarkers Prev. 2007 Highly methylated genes in colorectalneoplasia: implications for screening. 16:2686-96) and Li (NatBiotechnol. 2009 Sensitive digital quantification of DNA methylation inclinical samples. 27:858-63).

The subject method may be employed to diagnose patients with cancer or apre-cancerous condition (e.g., adenoma etc.), alone, or in combinationwith other clinical techniques (e.g., a physical examination, such as, acolonoscopy) or molecular techniques (e.g., immunohistochemicalanalysis). For example, results obtained from the subject assay may becombined with other information, e.g., information regarding themethylation status of other loci, information regarding the mutations atother loci, information regarding rearrangements or substitutions in thesame locus or at a different locus, cytogenetic information, informationregarding rearrangements, gene expression information or informationabout the length of telomeres, to provide an overall diagnosis of canceror other diseases.

In one embodiment, a sample may be collected from a patient at a firstlocation, e.g., in a clinical setting such as in a hospital or at adoctor's office, and the sample may be forwarded to a second location,e.g., a laboratory where it is processed and the above-described methodis performed to generate a report. A “report” as described herein, is anelectronic or tangible document which includes report elements thatprovide test results that may include a Ct value, or Cp value, or thelike that indicates the presence of mutant copies of the genomic locusin the sample. Once generated, the report may be forwarded to anotherlocation (which may the same location as the first location), where itmay be interpreted by a health professional (e.g., a clinician, alaboratory technician, or a physician such as an oncologist, surgeon,pathologist), as part of a clinical diagnosis.

Kits

Also provided are kits for practicing the subject method, as describedabove. The components of the kit may be present in separate containers,or multiple components may be present in a single container. Thecomponents of the kit may include: a) a first primer and a secondprimer, where the first primer corresponds to a methylated sequence inthe genomic locus and optionally contains a 3′ terminal nucleotide thatbase pairs with a methylated cytosine or its complement in themethylated sequence; and b) flap assay reagents comprising a flapendonuclease, a FRET cassette and, if the first primer does not containa 3′ terminal nucleotide that base pairs with a methylated cytosine inthe methylated sequence, an invasive oligonucleotide having a 3′terminal nucleotide that base pairs with a G or C residue thatcorresponds to a site of methylation in the genomic locus. Theparticulars of these reagents are described above. The kit may furthercontain an agent that modifies unmethylated cytosine to uracil. The kitfurther comprises PCR and flap reagents for amplification and detectionof a control nucleic acid.

In addition to the above-mentioned components, the kit may furtherinclude instructions for using the components of the kit to practice thesubject methods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate. In addition to theinstructions, the kits may also include one or more control samples,e.g., positive or negative controls analytes for use in testing the kit.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Example 1

Detection of Methylated C6ORF150 Sequences in the Presence ofUnmethylated C6ORF150

The assay was designed to detect and quantitate the methylated CpGsequences in the presence of unmethylated sequence. In order to simulatethe methylated and unmethylated genomic DNA, plasmids were prepared andcloned to match the sequence that results following the bisulfitereaction conversion of unmethylated C to U, which behaves as if it werea T in the PCR process, as exemplified for the vimentin sequence in FIG.3 . For each example, the methylated version of the sequence uses aplasmid with the CG motif intact and the unmethylated representativeplasmid replaces this with a TG motif.

In this example, 2 CGs were designed on each primer, and they were notat 3′ends of the primers. The assay was then used to detect methylatedcopies spiked in unmethylated copies at 4 different levels, including10⁴ methylated copies in 10⁵ unmethylated copies (1:10), 10³ methylatedcopies in 10⁵ unmethylated copies (1:100), 10² methylated copies in 10⁵unmethylated copies (1:1000), and 10 methylated copies in 10⁵unmethylated copies (1:10000).

The target sequence of the plasmid representing the methylated sequencewas as follows, with every C base corresponding to a methyl C for ananalogous genomic DNA:

(SEQ ID NO: 1) ATGGAATGTTAGGGGCGTTTCGATGGATTTTATCGAGTTTTCGGTTGTTTTCGAGGTCGTTTTGTTTAAGGCGGGAAAGTTCGGTTTCGTTAGGAAGTCGGGATTTCGGTAGAAAAAGAGCGTTTCGGATATTTAGGAGAGGTCGTTCGTTCGCGTAATTGGGGTTCGCGTTAAAAAGGTTTTTTAGCGCGTTTAGGATA CGTAGTC

The assay employed a forward primer 5′-GGGATTTCGGTAGAAAAAGAGCGT-3′ (SEQID NO:2), a reverse primer 5′-ACCTTTTTAACGCGAACCCCA-3′ (SEQ ID NO:3), aninvasive oligonucleotide, that was not the forward PCR primer5′-TCGGATATTTAGGAGAGGTg-3′ (SEQ ID NO:4), and a flap probe5′-GACGCGGAGCGTTCGTTCGCG-3′/3C6/(SEQ ID NO:5) where the areacorresponding to methylated bases is shown underlined and the 3′-end isblocked with a hexanediol group in order to inhibit primer extension.The first nine bases of the flap probe are the region cleaved away bythe flap endonuclease and then bind to the FRET cassette. Note that the3′-end base of the invasive probe, shown as a lower case g, is designedto mismatch the target so as to discourage primer extension by the Taqpolymerase. Primers, invasive oligos, and flap probes were supplied asnon-catalog items by Integrated DNA Technologies (IDT, Coralville,Iowa). The binding regions for the primers and invasive probe are shownon the target sequence underlined, and the target binding region of theflap probe is shown in italics:

(SEQ ID NO: 1) ATGGAATGTTAGGGGCGTTTCGATGGATTTTATCGAGTTTTCGGTTGTTTTCGAGGTCGTTTTGTTTAAGGCGGGAAAGTTCGGTTTCGTTAGGAAGTCGGGATTTCGGTAGAAAAAGAGCGTTTCGGATATTTAGGAGAGGT CGTTCGTTCGCGTAATTGGGGTTCGCGTTAAAAAGGTTTTTTAGCGCGTTTAGGATA CGTAGTC.

The FRET cassette used was 5′-FAM/TCT/Quencher/AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-3′/3C6 (SEQ ID NO:6), where FAM is fluorescein, thequencher is the Eclipse® Dark Quencher, and the 3′-end is blocked with ahexanediol group in order to inhibit primer extension. The FRET cassettewas supplied by Hologic (Madison, Wisconsin).

Cycling conditions were 95° C. for 3 min; 50 cycles at 95° C. for 20sec, 50° C. for 1 mM, and 70° C. for 30 sec, with a final 40° C. hold.Fluorescent signal acquisition was done at the 50° C. point in thecycle. The PCR reactions were done in LightCycler® 480 Multiwell 96Plates (Roche, Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂,and 250 μM dNTPs (Promega, Madison, Wisconsin). Taq polymerase was theiTaq enzyme (BioRad, Hercules, California) and the cleavage enzyme wasCleavase 2.0 (Hologic, Madison, Wisconsin). Forward primer concentrationwas 500 nM, reverse primer concentration was 500 nM, flap probe was at500 nM, invasive oligo probe was at 70 nM, and the FRET cassette wasused at a final concentration of 200 nM. All amplification and detectionwas performed in the LightCycler 480 optical thermocycler (Roche,Indianapolis, Indiana).

Data showing kinetic amplification curves and the crossing point, Cp, ofthe different ratios of mutant to wild type in the amplification samplesare shown in FIG. 5 . In these assays, the Cp is calculated as being thepoint at which fluorescence rose to 18% of the maximum fluorescence.

The design of primers, invasive probe, and flap probe used in thisexample was unable to detect 10² methylated copies in 10⁵ unmethylatedcopies (1:1000; FIG. 5 ). The reactions for detecting 10³ methylatedcopies in 10⁵ unmethylated copies (1:100) and 10⁴ methylated copies in10⁵ unmethylated copies (1:10) were also suppressed by excessive amountsof unmethylated gene copies (FIG. 5 ).

Example 2

Detection of methylated ZNF804B sequences in the presence ofunmethylated ZNF804B The assay was designed to detect and quantify themethylated CpG sequences of ZNF804B in the presence of unmethylatedZNF804B sequence. In order to simulate the methylated and unmethylatedgenomic DNA, plasmids were prepared and cloned to match the sequencethat results following the bisulfite reaction conversion of unmethylatedC to U, which behaves as if it were a T in the PCR process, asexemplified for the vimentin sequence in FIG. 3 . For each example, themethylated version of the sequence uses a plasmid with the CG motifintact and the unmethylated representative plasmid replaces this with aTG motif.

In this example, 1 CG was designed on each primer, and they were not at3′ends of the primers. The assay was then used to detect methylatedcopies spiked in unmethylated copies at 4 different levels, as inExample 1, including 10⁴ methylated copies in 10⁵ unmethylated copies(1:10), 10³ methylated copies in 10⁵ unmethylated copies (1:100), 10²methylated copies in 10⁵ unmethylated copies (1:1000), and 10 methylatedcopies in 10⁵ unmethylated copies (1:10000).

The target sequence of the plasmid representing the methylated sequencewas as follows, with every C base corresponding to a methyl C for ananalogous genomic DNA:

(SEQ ID NO: 7) TTAATTTGTTTGTTTTATTTGTGGTTGTATAGTTTATTTTTGTAATCGGTTGGGGAGTTGTTGTTTTTGTTAACGTCGTCGTTAGTTAGAGCGTTGAAGAAAAGTTGAAGGTTAGTAGGTAACGAAAGAGTAAAGA

The assay employed a forward primer 5′-GTGGTTGTATAGTTTATTTTTGTAATCGGT-3′(SEQ ID NO:8), a reverse primer 5′-ACCTTCAACTTTTCTTCAACGCTC-3′ (SEQ IDNO:9), an invasive oligonucleotide, that was not the forward PCR primer5′-GGGAGTTGTTGTTTTTGTTAAg-3′ (SEQ ID NO:10), and a flap probe5′-GACGCGGAGCGTCGTCGTTAG-3′/3C6/(SEQ ID NO:11) where the areacorresponding to methylated bases is shown underlined and the 3′-end isblocked with a hexanediol group in order to inhibit primer extension.The first nine bases of the flap probe are the region cleaved away bythe flap endonuclease and then bind to the FRET cassette. Note that the3′-end base of the invasive probe, shown as a lower case g, is designedto mismatch the target so as to discourage primer extension by the Taqpolymerase. Primers, invasive oligos, and flap probes were supplied asnon-catalog items by Integrated DNA Technologies (IDT, Coralville,Iowa).

The binding regions for the primers and invasive probe are shown on thetarget sequence underlined, and the target binding region of the flapprobe is shown in italics:

(SEQ ID NO: 7) TTAATTTGTTTGTTTTATTTGTGGTTGTATAGTTTATTTTTGTAATCGGTTGGGGAGTTGTTGTTTTTGTTAA CGTCGTCGTTAGTTAGAGCGTTGAAGAAAAGTTGAAGGTTAGTAGGTAACGAAAGAGTAAAGA.

The FRET cassette used was 5′-FAM/TCT/Quencher/AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-3′/3C6 (SEQ ID NO:6), where FAM is fluorescein, thequencher is the Eclipse® Dark Quencher, and the 3′-end is blocked with ahexanediol group in order to inhibit primer extension. The FRET cassettewas supplied by Hologic (Madison, Wisconsin).

Cycling conditions were 95° C. for 3 min; 50 cycles at 95° C. for 20sec, 50° C. for 1 min, and 70° C. for 30 sec, with a final 40° C. hold.Fluorescent signal acquisition was done at the point in the cycle. ThePCR reactions were done in LightCycler® 480 Multiwell 96 Plates (Roche,Indianapolis) in 10 mM MOPS pH 7.5, with 7.5 mM MgCl₂, and 250 μM dNTPs(Promega, Madison, Wisconsin). Taq polymerase was the iTaq enzyme(BioRad, Hercules, California) and the cleavage enzyme was Cleavase 2.0(Hologic, Madison, Wisconsin). Forward primer concentration was 500 nM,reverse primer concentration was 500 nM, flap probe was at 500 nM,invasive oligo probe was at 70 nM, and the FRET cassette was used at afinal concentration of 200 nM. All amplification and detection wasperformed in the LightCycler 480 optical thermocycler (Roche,Indianapolis, Indiana).

Data showing kinetic amplification curves and the crossing point, Cp, ofthe different ratios of mutant to wild type in the amplification samplesare shown in FIG. 6 . In these assays, the Cp is calculated as being thepoint at which fluorescence rose to 18% of the maximum fluorescence.

The design of primers, invasive probe, and flap probe used in thisexample could not detect 10 methylated copies in 10⁵ unmethylated copies(1:10000; FIG. 6 ). The reactions for detecting 10² methylated copies in10⁵ unmethylated copies (1:1000), 10³ methylated copies in 10⁵unmethylated copies (1:100), and 10⁴ methylated copies in 10⁵unmethylated copies (1:10) were also suppressed by excessive amounts ofunmethylated gene copies (FIG. 6 ).

Example 3

Detection of Methylated Vimentin Sequences in the Presence ofUnmethylated Vimentin

The assay was designed to detect and quantitate the methylated CpGsequences of vimentin (VIM) in the presence of unmethylated VIMsequence. In order to simulate the methylated and unmethylated genomicDNA, plasmids were prepared and cloned to match the sequence thatresults following the bisulfite reaction conversion of unmethylated C toU, which behaves as if it were a T in the PCR process, as shown for thevimentin sequence in FIG. 3 . The methylated version of the sequenceuses a plasmid with the CG motif intact and the unmethylatedrepresentative plasmid replaces this with a TG motif.

In this example, 3 CGs were designed on each primer of the vimentinmethylation detection assay, with one at the 3′ end of the forwardprimer. In this assay, the forward primer is also the invasiveoligonucleotide. There are also CG motifs located at the cleavage pointof the flap probe, in both senses. The assay was then used to detectmethylated copies spiked in unmethylated copies at 4 different levels,as in Example 1 and 2, including 10⁴ methylated copies in 10⁵unmethylated copies (1:10), 10³ methylated copies in 10⁵ unmethylatedcopies (1:100), 10² methylated copies in 10⁵ unmethylated copies(1:1000), and methylated copies in 10⁵ unmethylated copies (1:10000).

The target sequence of the plasmid representing the methylated sequencewas as follows, with every C base corresponding to a methyl C for ananalogous genomic DNA:

(SEQ ID NO: 12) TCGTGTTTTCGTTTTTTTATCGTAGGATGTTCGGCGGTTCGGGTATCGCGAGTCGGTCGAGTTTTAGTCGGAGTTACGTGATTACGTTTATTCGTATTTA TAGTTTGGGCGACG

The assay employed a forward primer 5′-GGCGGTTCGGGTATCG-3′ (SEQ IDNO:13), a reverse primer 5′-CGTAATCACGTAACTCCGACT-3′ (SEQ ID NO:14), anda flap probe 5′-GACGCGGAGGCGAGTCGGTCG-3′/3C6/(SEQ ID NO:15) where thearea corresponding to methylated bases is shown underlined and the3′-end is blocked with a hexanediol group in order to inhibit primerextension. The first nine bases of the flap probe are the region cleavedaway by the flap endonuclease and then bind to the FRET cassette.Primers and flap probes were supplied as non-catalog items by IntegratedDNA Technologies (IDT, Coralville, Iowa).

The binding regions for the primers and invasive probe are shown on thetarget sequence underlined, and the target binding region of the flapprobe is shown in italics:

(SEQ ID NO: 12) TCGTGTTTTCGTTTTTTTATCGTAGGATGTTCGGCGGTTCGGGTATCG CGAGTCGGTCGAGTTTTAGTCGGAGTTACGTGATTACGTTTATTCGTATTTA TAGTTTGGGCGACG.

The FRET cassette used was 5′-FAM/TCT/Quencher/AGCCGGTTTTCCGGCTGAGACTCCGCGTCCGT-3′/3C6 (SEQ ID NO:6), where FAM is fluorescein, thequencher is the Eclipse® Dark Quencher, and the 3′-end is blocked with ahexanediol group in order to inhibit primer extension. The FRET cassettewas supplied by Hologic (Madison, Wisconsin).

Cycling conditions were 95° C. for 2 min; 45 cycles at 95° C. for 20sec, 53° C. for 1 mi; and 40° C. to hold. Fluorescent signal acquisitionwas done at the 53° C. point in the cycle. The PCR reactions were donein LightCycler® 480 Multiwell 96 Plates (Roche, Indianapolis) in 10 mMMOPS pH 7.5, with 7.5 mM MgCl₂, and 250 μM dNTPs (Promega, Madison,Wisconsin). Taq polymerase was the HotStart GoTaq enzyme (Promega,Madison, Wisconsin) and the cleavage enzyme was Cleavase 2.0 (Hologic,Madison, Wisconsin). Forward primer concentration was 500 nM, reverseprimer concentration was 500 nM, flap probe was at 500 nM, and the FRETcassette was used at a final concentration of 200 nM. All amplificationand detection was performed in the LightCycler 480 optical thermocycler(Roche, Indianapolis, Indiana).

Data showing kinetic amplification curves and the crossing point, Cp, ofthe different ratios of mutant to wild type in the amplification samplesare shown in FIG. 7 . In these assays, the Cp is calculated as being thepoint at which fluorescence rose to 18% of the maximum fluorescence.

The design of primers, invasive probe, and flap probe used in thisexample could linearly detect down to 10 methylated copies in 10⁵unmethylated copies (1:10000) and is clearly superior (FIG. 7 ) to theperformance of Examples 1 and 2 (FIGS. 5 and 6 ).

1.-25. (canceled)
 26. A method for detecting methylation status of atarget genomic locus, the method comprising: 1) amplifying a targetgenomic locus in a reaction mixture comprising: a) a treated nucleicacid sample; b) amplification reagents comprising a thermostablepolymerase, nucleotides, a first primer and a second primer foramplifying the target genomic locus, wherein: i. the first primerhybridizes to a methylated sequence in the locus and contains a 3′terminal G or C nucleotide that corresponds to a methylated cytosine inthe genomic locus; and ii. the reagents preferentially amplifymethylated copies of the genomic locus, to produce an amplified sample;2) detecting the presence of amplified methylated copies of the genomiclocus in the amplified sample using a flap assay that employs flap assayreagents comprising a flap endonuclease, a FRET cassette, and a flapoligonucleotide that comprises a G or C nucleotide at a position thatcorresponds to the methylated cytosine and wherein an invasiveoligonucleotide distinct from the first primer is not included andwherein the first primer serves as an invasive oligonucleotide.
 27. Themethod of claim 26, wherein the flap oligonucleotide comprises aninternal G or C nucleotide at a position that corresponds to a secondmethylated cytosine in the genomic locus.
 28. The method of claim 26,wherein the first primer comprises an internal G or C nucleotide at aposition that corresponds to a second methylated cytosine in the genomiclocus.
 29. The method of claim 26, wherein the first and second primersboth hybridize to a methylated sequence in the genomic locus.
 30. Themethod of claim 26, wherein the nucleic acid sample comprises at least100 times more unmethylated copies of the genomic locus than methylatedcopies of the genomic locus.
 31. The method of claim 26, furthercomprising normalizing the amount of the amplified methylated copies ofthe genomic locus in the amplified sample relative to the amount of acontrol nucleic acid present in the nucleic acid sample, therebydetermining the amount of methylated copies of the genomic locus in thenucleic acid sample.
 32. The method of claim 31, wherein the controlnucleic acid is a locus different from the genomic locus.
 33. The methodof claim 31, wherein the control nucleic acid is detected using a flapassay that employs an invasive oligonucleotide having a 3′ terminalnucleotide that base pairs with an A or T residue at the site of themethylated cytosine, thereby detecting the presence of unmethlyatedcopies of the genomic locus.
 34. The method of claim 31, wherein theflap assay employs first flap assay reagents that include a firstinvasive oligonucleotide, a first flap probe having a first flap and afirst FRET cassette, and wherein the control nucleic acid is detectedusing second flap assay reagents that include a second invasiveoligonucleotide, a second flap probe having a second flap and a secondFRET cassette that produces a signal that is distinguishable from thefirst FRET cassette, wherein the first and second flap reagents are insame reaction mix.
 35. The method of claim 26, wherein methylation ofthe locus is cancer-related.
 36. The method of claim 26, wherein thelocus is that of BMP3, TFPI1, NDRG4, Septin 9, TFPI2, or Vimentin. 37.The method of claim 26, wherein the sample is obtained from a human. 38.The method of claim 37, wherein the sample is stool.
 39. The method ofclaim 26, wherein the amplifying and detecting steps are done using areaction mix that contains both PCR reagents and flap reagents, and noadditional reagents are added to the reaction mix between the amplifyingand detecting steps.
 40. The method of claim 39, wherein the reactionmix further comprises PCR reagents and flap reagents for amplifying anddetecting a second genomic locus.
 41. The method of claim 26, whereinthe target genomic locus is Vimentin.
 42. The method of claim 41,wherein the methylated sequence in the locus comprises the nucleic acidsequence of SEQ ID NO:12.
 43. The method of claim 41, wherein the firstprimer comprises the sequence of SEQ ID NO:13.
 44. The method of claim41, wherein the second primer comprises the sequence of SEQ ID NO:14.45. The method of claim 41, wherein the flap oligonucleotide comprisesthe sequence of SEQ ID NO:15.